DESCRIPTION (Adapted from the applicants Abstract): Legionella pneumophila, the agent of Legionnaire=s disease, is a major cause of both community-acquired and nosocomial pneumonia and can account for up to 30% of all pneumonias. This bacterium is an intracellular parasite of both human alveolar macrophages and aquatic protozoa. We have discovered a locus (pilBCD) that plays a major role in L. pneumophila physiology and virulence. Most importantly, this locus encodes a protein (PilD) that is analogous to peptidases involved in type-IV pilus biogenesis and protein (type II) secretion in gram-negative bacteria. A mutation in pilD abolished pilus production, indicating that L. pneumophila expresses type-IV pili. Of greater significance, the pilD mutant, but not a pilin deficient strain, was dramatically impaired in its ability to infect amoebae and macrophages, indicating that type II secretion is required for intracellular growth. While studying L. pneumophila pilBCD expression, we discovered that the transcription of the pilBCD locus is regulated by temperature and that the expression of Legionella type-IV pili is modulated by both temperature and CO2. To further our understanding of both type II secretion and intracellular parasitism, we will I) determine how temperature, CO2, and growth phase modulated pilD expression and isolate the corresponding regulatory elements, ii) using genetic, immunologic , and biochemical screens, identify the PilD-dependent secreted proteins that promote intracellular infection, and iii) elucidate the intracellular trafficking events that are orchestrated by PilD and type II secreted proteins. The results of this investigation will not only enhance our understanding of legionellosis but will serve as a model for the study of other intracellular pathogens.